Dry solid medium for storage and analysis of genetic material

ABSTRACT

The present invention provides a dry solid medium for storing a sample of a genetic material, including RNA and DNA, in a form suitable for subsequent analysis. The dry solid medium of the invention includes a dry solid matrix and a composition including a protein denaturing agent, and a chelating agent. The dry solid medium can further include a component which functions in subsequent analysis of the genetic material using, for example, PCR, reverse transcriptase initiated PCR, LCR, RFLP, or genetic hybridization. A component for subsequent analysis includes, for example, a nucleotide sequences such as a primer, DNA or RNA probe, and a target sequence stabilizer. The dry solid medium can also include a retaining agent to enhance retention of a component for subsequent analysis. The invention also provides methods for using the dry solid medium of the invention. The dry solid media and methods of use thereof are particularly suited for use in automated systems.

RELATED TECHNOLOGY

The reader is advised of technology related to the present inventionwhich is disclosed in U.S. patent applications Ser. No. 08/480,135, U.S.Pat. No. 5,756,126, 08/320,041, abandoned, and Ser. No. 08/159,104, U.S.Pat. No. 5,496,562. U.S. Ser. No. 08/480,135, U.S. Pat. No. 5,756,126,and 08/320,041, abandoned, are continuation-in-part applications of U.S.Ser. No. 08/159,104, U.S. Pat. No. 5,496,562. The disclosures of U.S.Ser. No. 08/480,135, U.S. Pat. No. 5,756,126, 08/159,104, U.S. Pat. No.5,496,562, and 08/320,041, abandoned, are incorporated herein byreference.

FIELD OF THE INVENTION

The field of the present invention pertains to a dry solid medium andmethod for collection of genetic material in a form suitable for storageor subsequent analysis. The invention further provides for analysis ofstored genetic material including methods suited for automated analyzingsystems.

BACKGROUND

Blood containing genetic material (including DNA or RNA) to be analyzedhas typically been transported from the place of removal, from a humanor animal, to the place of analysis as purified genetic material, liquidwhole blood, frozen whole blood or whole blood dried onto paper.However, all of these methods have disadvantages. Transport of geneticmaterial in blood as dried, purified genetic material is most desirable,but it requires a high standard of technical assistance to be availableat the place of removal from the human or animal. When technicalassistance is not available at the place of removal, whole blood orother unpurified samples are usually sent to a central facility wherethe genetic material is purified.

Transport of liquid whole blood often involves the need for sterility ofcollection. Under some circumstances, this is extremely inconvenient,for example, where the sample is a heel-prick taken from an infant. Thetransport of liquid whole blood or frozen blood may also requiretemperature controlled transport or a transport system other than theregular postal system. This is true even before considering publichealth concerns. In addition, concern over pathogens associated withwhole blood, such as the HIV virus, generally rule out the transport ofa potentially infectious liquid or frozen sample except under proper andexpensive supervision.

Transport of blood dried on filter paper is a proven alternative to theabove procedures. Genetic material can be extracted and isolated fromwhole blood spots which are dried on filter paper, in a form and insufficient quantities for use in genetic analysis. McCabe, E. R. B., etal., "DNA Microextraction From Blood Spots on Filter Paper Blotters:Potential Screening Applications to Newborn Screening," Hum. Genet.75:213-216 (1987). However, the procedure still suffers from a number ofdisadvantages. For example, typically, there has been no deliberate andrapid destruction of blood associated microorganisms. The presence ofpathogenic microorganisms may create a potential hazard for bloodhandling personnel. Moreover, some microorganisms may cause damage tothe genetic material. While some inhibition of microorganisms may occurthrough desiccation, it is known that slow desiccation, or even a smalldegree of rehydration under conditions of high relative humidity, mayallow the growth of DNA or RNA destroying microflora.

Another disadvantage of present methods for dry transport of geneticmaterial in blood is a lack of deliberate inhibition of processes whichdegrade genetic material. Hence, even in the presence of abacteriostatic agent there are conditions that permit enzymatic,nonenzymatic and autolytic breakdown of the genetic material.Furthermore, using presently available methods of dry storage,considerable difficulty is encountered if desorption of high molecularweight DNA or RNA is required. The surface adsorption affects can causeloss of genetic material which may cause the preferential loss of theleast degraded, i.e., the most desired class of DNA or RNA molecules.

Thus, there is a need for a safe, convenient and minimally laborintensive means for storage of a genetic material which is contained ina liquid sample.

However, even if a sample of genetic material is collected in a safe,convenient and reliable form for storage, subsequent analysis of thestored sample may give rise to logistic problems. This is especiallytrue when there are many different types of analyses to be performed ona collected sample. The logistics tend to become even more complex whenmultiple samples are submitted for analysis.

For example, some methods of analysis of genetic material for a specificgenetic sequence, such as polymerase chain reaction (PCR) analysis,require use of an oligonucleotide primer-pair. Generally a differentprimer-pair is used for each sequence analysis to be performed. And,because there are an extremely large number of possible primer pairs(for example, one for each sequence within the genes of humans, animalsand all other living organisms including the pathogens of humans andanimals), multiple sequence analyses in a single sample may give rise toimmense logistic problems. The problems are potentially greater whenmultiple samples of genetic material are received for analysis, such asat a centralized analyzing facility.

Automated analysis of genetic material provides enhanced efficiency forprocessing large numbers of samples. However, automated analysis of asample of genetic material still requires the automated system to haveseparate delivery devices for each different set of primers to preventthe occurrence of cross-contamination. Therefore, the range of geneticsequence analyses which can be performed at one time may be restricteddue to the inherent problem of cross contamination of primers. Hence,there is a need for reducing the restrictions against analyzing multiplesequences or multiple samples of genetic material using automatedsystems.

SUMMARY OF THE INVENTION

The present invention provides a safe, convenient and minimally laborintensive apparatus and method for storage or analysis of a geneticmaterial contained in a liquid medium. The invention further providesfor storage of genetic material in such a way as to allow for simplifiedanalysis of one or more samples of genetic material using automatedsystems.

The invention includes a dry solid medium for storage of a sample ofgenetic material. The dry solid medium of the invention is composed of asolid matrix and a composition which when applied to the dry solidmedium protects against degradation of genetic material stored on thedry solid medium. The dry solid medium may further provide inactivationof microorganisms, including those which may be pathogenic to humans.

The composition of the dry solid medium may include a weak base, achelating agent and a protein denaturant such as a detergent. In anotherembodiment, if long-term storage of a sample of genetic material isdesired, the composition may further include a free radical trap.

According to the invention, genetic material stored on the dry solidmedium may be analyzed using methods known in the art, for example,polymerase chain reaction (PCR), ligase chain reaction (LCR), reversetranscriptase initiated PCR, DNA or RNA hybridization techniquesincluding restriction fragment length polymorphism (RFLP) and othertechniques using genetic or DNA or RNA probes, genomic sequencing,enzymic assays, affinity labeling, methods of detection using labels orantibodies and other similar methods.

Genetic material stored on the dry solid medium of the invention may beanalyzed in situ or after removal from the dry solid medium.

In another embodiment of the invention, the dry solid medium of theinvention may include a component which is functional in subsequentanalysis of the stored sample of genetic material. According to thisembodiment, a component for subsequent analysis includes, for example,nucleotide sequences such as PCR primers, target sequence stabilizers,genetic probes, primers for genetic sequencing or sets ofoligonucleotide substrates for LCR analysis. As disclosed herein, thecompound for subsequent analysis may also include a suitably stableenzyme.

The invention also provides methods for using the dry solid medium. Inone embodiment, the invention provides for storing a sample of geneticmaterial on the dry solid medium in a substantially nondegraded form.The invention further provides a method for storage and subsequentanalysis of a stored sample of genetic material. In a preferredembodiment, prior to analysis, the stored genetic material may be washedto remove compounds which may be associated with a sample of geneticmaterial and which may inhibit subsequent analysis. Such compoundsinclude, for example, protein, hemoglobin and components of thecomposition of the dry solid medium. The invention provides for thestored genetic material to be washed using an aqueous or a nonaqueouswash system.

The aqueous or nonaqueous wash system used to wash a sample of geneticmaterial preferably provide for removing compounds which may inhibitsubsequent analysis, without substantially affecting the stored geneticmaterial or a component for subsequent analysis which may be included onthe dry solid medium. Moreover, the aqueous and nonaqueous wash systemsof the invention may also be used to wash genetic material which isstored, for example, on a solid matrix which does not have sorbed to itthe composition of the invention.

In one embodiment, the nonaqueous wash system of the invention providesfor contacting a sample of genetic material stored on the dry solidmedium of the invention with a herein described single phase phenolsolution. The single phase phenol solution is removed and the dry solidmedium containing a sample of genetic material is subsequently contactedwith an aqueous alcohol wash solution. The aqueous alcohol wash solutionis removed and the sample of genetic material may further be washed withan aqueous ionic solution. The washed sample of genetic material maythen be analyzed by methods known in the art, including those mentionedabove.

Aqueous wash systems are known in the art. Under some circumstances, awash system may cause some loss of a component for subsequent analysiswhich may be included on the dry solid medium of the invention. In oneembodiment of the invention, a herein disclosed retaining agent may beused to reduce loss of a component for subsequent analysis duringprocessing of the sample of genetic material.

A sample of genetic material stored on a dry solid medium of theinvention may also be analyzed using standard enzymic tests used, forexample, to detect phenylketonuria (PKU) or galactosemia, preferablyafter neutralizing the effects of protein denaturing components of thedry solid medium using, for example, a herein disclosed convertersolution.

The dry solid medium and the dry solid medium including a component forsubsequent analysis are particularly useful for analysis of a sample ofgenetic material using automated systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Photograph of ethidium bromide stained gel of PCR amplified DNAfrom blood samples stored on a dry solid medium of the invention.

FIG. 2, Photograph of ethidium bromide stained gel of PCR amplified DNAfrom blood samples stored on a dry solid medium of the invention whichincluded oligonucleotide primers for PCR analysis.

FIG. 3, Photograph of ethidium bromide stained gel of Eco R1 digestionof DNA from human blood stored on the dry solid medium of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a dry solid medium for collection ofgenetic material in a form suitable for storage or subsequent analysis.The invention further provides methods for subsequent analysis of thegenetic material collected on the dry solid medium of the invention. Thedry solid medium and methods of use, disclosed herein, generally providea safe, convenient and reliable storage medium for samples of geneticmaterial which allows for accurate analytical results. Moreover, theinvention further provides for enhanced analytical efficiency whenmultiple samples are analyzed using automated systems, for example, at acentralized analyzing facility.

In several places throughout the present specification guidance isprovided through lists of examples. The inventor wishes to make clearthat in each instance, the recited list serves only as a representativegroup. It is not meant, however, that the listed examples are exclusive.

As used herein, the phrase "genetic material" (GM) means either or bothdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to theinvention, a sample of GM is collected on a dry solid medium of theinvention by removing the sample from a source and applying the sampleto a herein described dry solid medium. Methods for removing a sample ofgenetic material from a source are known in the art. For example, asample of genetic material in blood may be removed from a human oranimal source by venipuncture and the removed sample then applied to thedry solid medium of the invention.

As used herein a "sample of genetic material" or "sample of GM" includesa liquid having dissolved, suspended, mixed or otherwise containedtherein, either or both DNA or RNA, cells which contain either or bothDNA or RNA or cell components which contain either or both DNA or RNA.Once the sample of GM is applied to a dry solid medium, the liquid tendsto evaporate (evaporation may be enhanced by drying methods known in theart, for example, a warm air dryer) leaving the DNA or RNA entrained tothe dry solid medium in a dry form. The GM entrained to the dry solidmedium in "dry form" may be purified DNA or RNA, semipurified DNA or RNAor DNA or RNA in cells.

The sample of GM which is applied to a dry solid medium may be derivedfrom any source. This includes, for example, physiological/pathologicalbody liquids (e.g., secretions, excretions, exudates and transudates) orcell suspensions (e.g., blood, lymph, synovial fluid, semen, salivacontaining buccal cells, skin scrapings, hair root cells, etc.) ofhumans and animals; physiological/pathological liquids or cellsuspensions of plants; liquid products, extracts or suspensions ofbacteria, fungi, plasmids, viruses etc.; liquid products, extracts orsuspensions of parasites including helminths, protozoas, spirochetes,etc.; liquid extracts or homogenates of human or animal body tissues(e.g., bone, liver, kidney, etc.); media from DNA or RNA synthesis;mixtures of chemically or biochemically synthesized DNA or RNA; and anyother source in which DNA or RNA is or can be in a liquid medium.

A dry solid medium of the invention provides for storage or subsequentanalysis of a sample of GM. A dry solid medium is composed of a solidmatrix having sorbed thereto a composition which can protect againstdegradation of the GM stored on the medium. The composition may alsocause inactivation of microorganisms. This includes microorganismsassociated with a sample of GM which may potentially degrade the sampleof GM or may be potentially pathogenic to human handlers of the storedsample of GM.

A dry solid medium and a composition sorbed to a solid matrix isdisclosed in allowed U.S. patent application Ser. No. 08/159,104, U.S.Pat. No. 5,496,562, which is incorporated herein by reference.

As used herein, the term "storing", "storage", "stored" and otherderivatives of the word "store" means the preservation of GM in a formsuitable for subsequent analysis or which has not undergone substantialdegradation. The time period for which GM may be stored according to theinvention may be as short as the time necessary to transfer a sample ofGM from a collection source to the place where subsequent analysis is tobe performed. The conditions under which a sample of GM may be stored ona dry solid medium of the invention is variable. Typically, samples arestored at temperatures from -200° C. to 40° C. In addition, storedsamples may optionally be stored in dry or desiccated conditions orunder an inert atmosphere. Storage may be for a few seconds up to manyyears, preferably, about a few seconds up to 100 years.

In another embodiment of the invention, a dry solid medium may alsoinclude a component which is functional in a subsequent analysis to beperformed on a stored sample of GM. Subsequent analysis which may beperformed on a sample of GM stored on the dry solid medium includeanalysis methods known in the art, for example, polymerase chainreaction (PCR), ligase chain reaction (LCR), reverse transcriptaseinitiated PCR, DNA or RNA hybridization techniques including restrictionfragment length polymorphism (RFLP) and other techniques using geneticprobes (DNA or RNA), genomic sequencing, enzymatic assays, affinitylabeling, methods of detection using labels or antibodies and othersimilar methods. In addition, the inventor recognizes that many newanalytical and diagnostic methods may be developed in the future forwhich the dry solid medium and method of the invention may be suitableand which would fall within the spirit and scope of the claims appendedhereto.

In the case of stored RNA, particularly unstable RNA, a component forsubsequent analysis which may be included on a dry solid medium mayprovide protection against RNA degradation. Such a component includesRNase inhibitors and inactivators, genetic probes, complementary DNA orRNA (or functionally equivalent compounds), proteins and organicmoieties that stabilize RNA or prevent its degradation.

Generally, a dry solid medium of the invention is also a suitable mediumfor storage of a component for subsequent analysis which may be includedon the dry solid medium.

Once the GM has been collected on the dry solid medium, it may beencased in a protective material, for example, a plastic film, which mayfurther protect against GM degradation during storage. Subsequentanalysis of the GM stored on the solid medium of the invention may beperformed in situ or, alternatively, the GM may first be removed fromthe dry solid medium prior to subsequent analysis.

I. THE DRY SOLID MEDIUM

A dry solid medium of the invention provides for entrainment of GM tothe dry solid medium. As used herein the term "entrain", and derivativesthereof means that during storage the GM is bound to the dry solidmedium without substantial reliance on ionic, covalent or van der waalsinteractions. As used herein, "without substantial reliance" means thatsuch interaction may not be a significant factor in maintaining the GMon the dry solid medium. Preferably, ionic, covalent or van der waalinteraction is not necessary for entrainment of GM to the dry solidmedium.

A dry solid medium of the invention includes a composition sorbed to asolid matrix. As used herein, the term "sorb" means that the compositionof the invention is absorbed, adsorbed or otherwise incorporated into oronto a solid matrix in such a way as not to be readily removed from thematrix unless subjected to conditions which are intentionally orinadvertently performed to remove the sorbed composition from the solidmatrix. Preferably the sorbed composition is not readily removed undernormal storage conditions.

A solid matrix suitable for a dry solid medium and method of theinvention includes any material to which the composition will sorb andwhich does not inhibit storage or subsequent analysis of the GM appliedto the dry solid medium. This includes a two-dimensional flat drymatrices or three-dimensional matrices such as a matrix combined with abinder to form a pellet or tablet to which the composition is sorbed.

In one preferred embodiment, the solid matrix is of a porous nature toprovide entrainment of the GM onto the dry solid medium. A solid matrixsuitable for this purpose includes, but is not limited to, a matrixwhich is cellulose based (e.g., cellulose, nitrocellulose orcarboxymethylcellulose papers), hydrophilic polymers including synthetichydrophilic polymers (e.g., polyester, polyamide, carbohydratepolymers), polytetrafluroethylene (Empore™, 3M, St. Paul, Minn.),fiberglass and porous ceramics.

According to the invention, GM may also be collected on a solid matrixwhich lacks the below-described composition of the invention. Inaddition, a component for subsequent analysis of a sample of GM may alsobe included on a solid matrix which lacks the composition of theinvention. Furthermore, hemoglobin, protein or other compound associatedwith a sample of GM may be removed from a sample of GM stored on a solidmatrix, which may or may not include a component for subsequent analysisof the stored GM, using the below described aqueous or nonaqueousextraction procedure. However, by using only a solid matrix for storageof GM, the GM degradation protection and microorganism inactivationeffects of the composition of the invention may not be present.

To prepare a dry solid medium of the invention, a composition whichprotects against degradation of GM is sorbed to the solid matrix. Asused herein, the phrase "protects against degradation of GM" means thata dry solid medium of the invention maintains the stored GM in asubstantially nondegraded form. This provides a sample of GM suitablefor many different types of subsequent analytical procedures. Protectionagainst degradation of GM may include protection against substantialdamaging of GM due to GM damaging events such as that caused by chemicalor biological agents including action of bacteria, free radicals,nucleases, ultraviolet radiation, oxidizing agents, alkylating agents,or acidic agents (e.g., pollutants in the atmosphere).

The composition sorbed to the solid matrix of the invention may includeone or more of a weak base, a chelating agent, or a protein denaturingagent such as a detergent or surfactant. In addition, the compositionsorbed to the dry solid medium may also include a free radicle trap.

As used herein, a "free radical trap" is a compound which issufficiently reactive to be preferred, over a DNA molecule or acomponent thereof, as a reactant with a free radical, and which issufficiently stable not to generate damaging free radicals itself.

As used herein, a "weak base" suitable for the composition of theinvention may be a Lewis base which has a pH of about 6 to 10,preferably about pH 8 to 9.5. One function of the weak base may be toact as a buffer to maintain a composition pH of about 6 to 10,preferably about pH 8.0 to 9.5, for example, pH 8.6. Hence, a weak basesuitable for the composition of the invention may, in conjunction withother components of the composition, provide a composition pH of 6 to10, preferably, about pH 8.0 to 9.5. Suitable weak bases according tothe invention include organic and inorganic bases. Suitable inorganicweak bases include, for example, an alkali metal carbonate, bicarbonate,phosphate or borate (e.g., sodium, lithium, or potassium carbonate).Suitable organic weak bases include, for example, tris-hydroxymethylamino methane (Tris), ethanolamine, triethanolamine and glycine andalkaline salts of organic acids (e.g., trisodium citrate). A preferredorganic weak base is a weak monovalent organic base, for example, Tris.The weak base may be either a free base or a salt, for example, acarbonate salt.

Although the inventors do not wish to be limited to a single theory, itis believed that the weak base may provide a variety of functions,including protecting the GM from degradation. In addition to providing abuffer system, it is also believed that the weak base can act to ensureproper action of the below described chelating agent in binding metalions. In addition, the weak base may also prevent the action of acidnucleases which may not be completely dependent on divalent metal ionsfor functioning.

The composition of the dry solid medium can also include a chelatingagent. As used herein, a chelating agent is any compound capable ofcomplexing multivalent ions including Group II and Group III multivalentmetal ions and transition metal ions (eg., Cu, Fe, Zn, Mn, etc).According to the invention, a preferred chelating agent is a strongchelating agent such as to ethylene diamine tetraacetic acid (EDTA).Chelating agents such as a citrate or oxalate are also suitable for theinvention. The chelating agent may be added to the compositionsimultaneous with application of a sample of GM to the dry solid medium.

Although the inventors do not wish to be limited to a particular theory,it is believed that one function of the chelating agent of the inventionis to bind multivalent ions which if present with the stored GM maypartake in causing damage to the GM. Ions which may be chelated by thechelating agent include multivalent active metal ions, for example,magnesium and calcium, and transition metal ions, for example, iron.Both calcium and magnesium are known to promote GM degradation by actingas co-factors for enzymes which may destroy GM (e.g., most knownnucleases). In addition, transition metal ions, such as iron, mayreadily undergo oxidation and reduction and damage nucleic acids by theproduction of free radicals or by direct oxidation.

The composition of the dry solid medium can further include a proteindenaturing agent. As used herein, a protein denaturing agent functionsto denature non-GM compounds, for example, proteins, which areassociated with the stored GM. If the protein denaturing agent is adetergent or a surfactant, the surfactant may also act as a wettingagent to facilitate the uptake of a sample by the dry solid medium. Theterms "surfactant" and "detergent" are synonymous and may be usedinterchangeably throughout the specification. According to theinvention, any agent which denatures proteins without substantiallyaffecting a sample of GM may be suitable for the invention. Preferredprotein denaturing agents include detergents. As used herein"detergents" include ionic detergents, preferably anionic detergents.

A preferred anionic detergent suitable for the invention may have ahydrocarbon moiety, such as an aliphatic or aromatic moiety, and one ormore anionic groups. Particularly preferred anionic detergents includesodium dodecyl sulphate (SDS) and sodium lauryl sarcosinate (SLS).

In one preferred embodiment, the ionic detergent of the invention causesinactivation of a microorganism which has protein or lipid in its outermembranes or capsids, for example, fungi, bacteria or viruses. Thisincludes microorganisms which may be pathogenic to humans or which maycause degradation of GM.

Although not wishing to be limited to a single theory, it is believedthat inactivation of a microorganism by a detergent is a result ofdestruction of the secondary structure of the organisms externalproteins, internal proteins, protein containing membranes, or any otherprotein necessary for viability. The inventor recognizes that thedetergent may not inactivate some forms of organisms, for example,highly resistant bacterial spores and extremely stable enteric virions.Moreover, the inventor further recognizes that while it may be desirableto inactivate a microorganism, the GM of a microorganism, eitherassociated with the stored sample of GM or not, is also amenable forstorage or subsequent analysis on the dry solid medium of the invention.

The composition of the invention may optionally include a free radicaltrap. A free radical trap according to the invention is defined above.Examples of a suitable free radical trap includes: uric acid or a uratesalt, mannitol, benzoate (Na, K, Li or tris salt), 1-3 dimethyl uricacid, guanidine, guanine, thymine, adenine, cytosine,N-acetyl-histidine, histidine, deferoxamine, dimethyl sulfoxide, 5'5'dimethyl pyrroline-N-oxide, thiocyanate salt and thiourea. Preferredfree radical traps include mannitol, thiocyanate salts, uric acid or aurate salt. According to the invention, the longer the period of timefor which the GM is to be stored the more likely that a free radicaltrap may be advantageously included in the composition sorbed to thesolid matrix. However, even if the GM is only to be stored for a matterof minutes, a free radical trap may still be incorporated into thecomposition.

While the inventor does not wish to be limited to any single theory, itis believed that one function of the free radical trap may be to trap GMdamaging free radicals. For example, when the free radical trap used isuric acid or urate salt it may be converted to allantoin which may alsoact as a free radical trap that preferentially accepts free radicalsthat would otherwise damage nucleotide bases, for example, guanine.

Preferably, the free radical trap reacts with free radicals regardlessof source (including free radicals present in the air). Free radicalsmay be generated through oxidation or reduction of iron in blood.Typically, free radicals are believed to be generated by spontaneousoxidation of the groups which are present, for example, in denaturedserum protein of blood. Free radicals may also be generated by radiationsuch as UV light, x-rays and high-energy particles.

In addition, free radical traps which are also a weak acid, e.g. uricacid, may also function as a component of the buffering system providedby the weak base discussed above. Also, as in the case of uric acid or aurate salt, the free radical trap may act as an erodible surface becauseit is sparingly soluble such that a GM sample dried onto its crystalswill be released as the urate beneath erodes. Hence, components of thecomposition, such as the free radical trap may enhance removal of astored sample of GM if in situ processing is not desired.

Furthermore, after the sample of GM is applied to a dry solid medium,the dry solid medium with the applied sample of GM may be encased in aprotective material, for example, a plastic film, which may furtherprotect against degradation of stored GM. Examples of plastic filmswhich are suitable according to the invention include polystyrene,polyethylene, polypropylene and other lamination plastics. Encasing thedry solid medium in a protective material may be accomplished by methodsknown in the art.

One simple method for encasing the dry solid medium in a plastic film isto put a dry solid medium into a container, e.g., a polyethylene bag,which is of sufficient size to hold the dry solid medium such that whena plastic film in liquid form is added to the container all parts of thedry solid medium will be coated by the liquid. The plastic film, inliquid form, is added to the container to coat the dry solid medium. Theliquid plastic film is allowed to dry to provide a plastic film coatingwhich encases the dry solid medium. Prior to analysis, the plastic filmis removed from the dry solid medium using methods known in the art, forexample, dissolving with organic solvents such as chloroform ormechanical stripping.

The inventors further note that a dry solid medium including componentsfor subsequent analysis (discussed below), with an applied sample of GM,may also be encased in a protective material as described above.

II. SUBSEQUENT ANALYSIS OF GENETIC MATERIAL STORED ON THE DRY SOLIDMEDIUM

As used herein, "subsequent analysis" includes any analysis which may beperformed on a sample of GM stored on a dry solid medium of theinvention. The GM stored on a dry solid medium may be analyzed in vitro.Alternatively, the GM may be removed from the dry solid medium prior toanalysis. The sample of GM may be subjected to chemical, biochemical orbiological analysis. Examples of subsequent analysis which may beperformed on samples of GM stored on the dry solid medium includepolymerase chain reaction (PCR), ligase chain reaction (LCR), reversetranscriptase initiated PCR, DNA or RNA hybridization techniquesincluding restriction fragment length polymorphism (RFLP) and othertechniques using genetic probes, genomic sequencing, enzymatic assays,affinity labeling, methods of detection using labels or antibodies andother similar methods.

In addition, diagnostic procedures presently performed on whole bloodsamples, for example, the Guthrie test for phenyl-ketonuria (PKU) orenzymatic tests, for example, galactosemia may be performed on bloodsamples stored on a dry solid medium of the invention after firstneutralizing the affects of protein denaturing components of the drysolid medium, for example, using a "converter solution."An example of aconverter solution is disclosed in Example 5.

GM stored on a dry solid medium of the invention may serve as a templatefor subsequent analysis of the GM using PCR methods. This includes PCRusing DNA or RNA as a template. When RNA is the GM to be analyzed, theRNA may serve as a template using reverse transcriptase initiated PCR.The DNA sequence produced from the RNA template may then serve as atemplate for further PCR amplification.

Compounds associated with a stored sample of GM, including hemoglobin,protein or components of the composition of the dry solid medium, mayinterfere with subsequent analysis of a sample of GM stored on the drysolid medium. Preferably, such interfering compounds are substantiallyremoved from the dry solid medium prior to subsequent analysis. If, forexample, the sample of stored GM is in whole blood, prior to analysis byPCR, LCR, RFLP, reverse transcriptase initiated PCR, genetic probing orother technique, preferably, hemoglobin or protein associated with theGM stored on the dry solid medium is preferably removed. Hemoglobin orprotein associated with the GM may be removed using, for example,aqueous or nonaqueous extraction procedures. In addition, an interferingcompound which is a component of the composition of the invention (e.g.,an ionic detergent) may be removed, using aqueous or nonaqueousextraction procedures or the herein disclosed converter solution, priorto subsequent analysis.

In one preferred embodiment, hemoglobin, blood protein or other proteinassociated with a sample of GM may be removed using nonaqueousextraction procedures, for example, the below-described single phasephenol wash method. In addition, the single phase phenol wash may befollowed with the below-described ionic aqueous alcohol wash to provideions necessary for subsequent PCR, LCR or similar analysis of GM storedon the dry solid medium. Methods for PCR, LCR, reverse transcriptaseinitiated PCR, DNA or RNA hybridization, genomic sequencing, and othermethods of subsequent analysis are known in the art.

In another embodiment of the invention, a sample of GM associated withprotein or hemoglobin which is stored on a dry solid medium of theinvention may be removed using aqueous extraction. One such aqueousextraction procedure, for example, uses aqueous SDS and mercaptoethanol.According to this procedure, the sample of GM stored on the dry solidmedium, with or without components for subsequent analysis, is washedwith an alkaline solution of aqueous 6% SDS at pH 8.5 to 9.0 whichcontains a thiol, for example, 20 mM 2-mercaptoethanol ordithiothreitol. Another such aqueous extraction procedure, for example,uses pure water. The GM is then washed with iso-propanol to removesubstantially all water, remaining impurities, and unwanted components(e.g., the iso-propanol wash may be accomplished using the aqueousalcohol wash solution described below for the single phase phenol wash).

As will be apparent to those skilled in the art, in situ subsequentanalysis of GM stored on a dry solid medium of the invention with orwithout components for subsequent analysis may be of particular benefitin automated systems. As used herein, an "automated system" includes an"automatic fluid delivery system." According to the invention, an"automatic fluid delivery system" includes hand-held and robotic fluiddelivery systems. Typically, an automatic fluid delivery system is adevice which dispenses and removes fluid reagents to and from individualwells of multi-well reaction plates. A hand-held automatic fluiddelivery system comprises a single plunger handle with multiple fluidaspirating and dispensing ends to simultaneously aspirate and dispensefluid of a fluid reaction system from single or multiple fluid reactionwells simultaneously. Robotic automatic fluid delivery systems arecomputer operated rather than hand-held and include such products as,for example, BIOMEK 2000 (Beckman Instruments, Fullerton, Calif.),Zymark Benchmate (Zymark, Hopkinton, Mass.) and ROSYS PLATO, Rapperswil,Switzerland. Automatic fluid delivery systems are also discussed in U.S.Ser. No. 08/320,041 which is incorporated herein by reference.

A. SINGLE PHASE PHENOL WASH

Another aspect of the present invention provides an organic extractionmethod for removing hemoglobin or protein associated with a storedsample of GM. According to the invention, hemoglobin or protein may beremoved from a stored sample of GM using a single phase phenol wash.Preferably, the single phase phenol wash also removes components of thecomposition of the invention (e.g., the detergent). The single phasephenol wash is performed by contacting a single phase phenol solutionwith a dry solid medium having applied thereto a sample of GM. Thesingle phase phenol wash is removed from the dry solid medium containinga sample of GM and the dry solid medium is then contacted with anaqueous alcohol wash. The aqueous alcohol wash is then removed. If thesample of GM is to be analyzed by PCR, LCR or other method which relieson ions, the sample of GM may then be contacted with an ionic aqueousalcohol wash prior to PCR analysis. A nonaqueous wash system such as thesingle phase phenol wash (described below) or an aqueous wash system(described above) may also be used to wash a sample of GM stored on asolid matrix lacking the composition of the invention and which may ormay not include a component for subsequent analysis of the GM.

The single phase phenol wash provides for removing a protein,hemoglobin, or a component of the composition of the dry solid mediumwhich may effect the subsequent treatment to be performed on the GMstored on the dry solid medium without effecting the stored GM. Unlikeprior art methods, the present wash system does not require two phaseseparations (aqueous and organic phase) during processing and tends notto dissolve GM present in blood. The single phase phenol wash is alsoamenable to automated systems.

The single phase phenol wash is also disclosed in allowed U.S. patentapplication Ser. No. 08/159,104, U.S. Pat. No. 5,496,562, and co-pendingSer. No. 08/480,135, U.S. Pat. No. 5,756,126 and 08/320,041, abandoned.The wash includes two solutions, referred to below as "single phasephenol solution" (solution A) and "aqueous alcohol wash solution"(solution B). A third solution, "ionic aqueous alcohol wash solution"(solution C), may be used, after the single phase phenol wash if thesample of GM is to be analyzed using a method which requires additionalions, for example, PCR and LCR.

Solution A: The single phase phenol solution includes phenol andhydroxyquinoline saturated with Tris-acetate and mercaptoethanol.Preferably, 50 grams (gm) of phenol is combined with 120 milligram (mg)of 8-hydroxyquinoline saturated with 10 milliliter (ml) of 1.0 molar (M)Tris-acetate at pH 6.0 to 9.0, preferably pH 8.0, and 1.0 ml2-mercaptoethanol. After saturation by shaking at a temperature of about0° C. to 20° C., preferably 0° C. to 4° C., the aqueous phase is removedand discarded. (The inventor notes that the single phase phenol wash isbelieved to be saturated with Tris Acetate and mercaptoethanol at 4° C.However, at use temperature, which is typically greater than 4° C., thesolution is not believed to be saturated.) Solution B: The aqueousalcohol wash solution utilizes any C₂ -C₅ alcohol, such as isopropanolor n-propanol, or any other similar water miscible solvent combined withan acetate salt or similar organic soluble salt which provides a pH ofabout 7.0 to 9.0, preferably about pH 7.8. In a preferred embodiment 75%by volume isopropanol is combined with 25% by volume of 0.1M potassiumacetate at a pH of about 7.8.

Solution C: The ionic aqueous wash solution is a wash solution whichprovides appropriate ions, for example, magnesium, manganese, sodium,protamine, tris and similar ions which may be useful in a subsequentanalysis procedure. For PCR analysis, magnesium is preferred.Accordingly, the ionic alcohol wash solution utilizes any C₂ -C₅alcohol, such as isopropanol and n-propanol, or any other similar watermiscible solvent combined with an acetate salt or similar organicsoluble salt which provides a pH of about 6.0 to 8.5, preferably aboutpH 7.8. In a preferred embodiment for magnesium ions, 75% by volumeisopropanol is combined with 25% by volume of 0.01M magnesium acetate ata pH of about 7.5.

According to the invention, a sample of GM associated with hemoglobin orprotein which is stored on the dry solid medium is contacted withsolution A for about 15 minutes to about 3 hours, preferably about 1.5to about 2 hours, at a temperature which is greater than 37° C.,preferably about 45-65° C., more preferably about 50° C. The temperatureat which the sample of GM is contacted with solution A is not criticalif the dry solid medium of the invention does not also include acomponent for subsequent analysis, such as a primer, which is discussedbelow. The solution A is then removed. Preferably, the sample of GM isthen quickly washed 1-4 times, for a few seconds each time with freshsolution A.

After removal of solution A, the sample of GM is washed, for a fewseconds, 1-4 times with solution B. Preferably, the solution B washesare at about 10° C. to 25° C. If solution C is to be used, aftersolution B is removed, solution C is applied to the sample of GM forabout 1-60 minutes, preferably about 30 minutes, at a temperature ofabout 10° C. to 25° C. The solution C is removed and the sample of GM onthe dry solid medium is dried.

Generally, the single phase phenol wash of the invention provides asubstantially protein or hemoglobin free GM that provides a veryconvenient starting point for a variety of analytical procedures basedon nucleic acids. The use of the single phase phenol wash and ionicaqueous alcohol wash is further described in the Examples.

According to the invention, the single phase phenol wash may be used toprovide a substantially protein or hemoglobin free sample of GM storedon any solid system. Moreover, as described below, the single phasephenol wash is of particular benefit when a solid storage systemincorporates a component for subsequent analysis such as anoligonucleotide primer. Hence, the single phase phenol wash is useful toremove proteins or hemoglobin from a sample of GM stored on a dry solidmedium regardless of whether or not the storage medium incorporates theGM protecting composition of the invention and regardless of whether ornot the storage medium includes components necessary for subsequentanalysis.

B. DRY SOLID MEDIUM INCLUDING COMPONENTS FOR SUBSEQUENT ANALYSIS OFGENETIC MATERIAL

In another embodiment of the invention, the dry solid medium of theinvention may include a component for subsequent analysis of a storedsample of GM. As used herein, "subsequent analysis" means any analysiswhich may be performed on a sample of GM stored on the dry solid medium.This includes chemical, biochemical or biological analysis. Examples ofsubsequent analysis which may be performed on a sample of GM stored on adry solid medium include PCR, LCR, reverse transcriptase initiated PCR,DNA or RNA hybridization techniques including RFLP and other techniquesusing genetic or DNA or RNA probes, genomic sequencing, enzymaticassays, affinity labeling, methods of detection using labels orantibodies and other methods known in the art.

As used herein, a "component for subsequent analysis" of a stored sampleof GM includes a molecule, compound, reagent or other component whichmay be functional in analyzing, or preserving for analysis, a sample ofGM, or analyzing some other component stored with the GM on the drysolid medium.

In some instances, subsequent analysis will involve an analytical methodwhich is used to establish the absence or presence of a particulargenetic sequence. The genetic sequence to be analyzed is termed a"target sequence." The target sequence or other region of a sample ofgenetic material may serve as a template for PCR amplification.

One or more components for subsequent analysis may be included on thedry solid medium. The component included on the dry solid medium isdependent on the type of analysis to be performed on the sample of GM.The advantage of including a component for subsequent analysis will bereadily apparent in situations where large numbers of samples of GM aresimultaneously analyzed using automated systems. A component forsubsequent analysis which may be included on a dry solid medium include,for example, an ion, an enzyme and a nucleotide sequence.

As used herein, "nucleotide sequence" includes compounds such as aprimer, (e.g., a PCR primer, a primer for reverse transcriptaseinitiated PCR), sets of oligonucleotide substrates for LCR, DNA or RNAprobe, oligonucleotides used in genetic sequencing, and a targetsequence stabilizer.

As used herein, a "primer" means one pair of oligonucleotides, each ofwhich is capable of annealing to a complementary nucleic acid sequencein a sample of GM, and which are oriented so that each primer will primethe synthesis of a strand of DNA (or conceivably, a strand of RNA)containing a sequence complimentary to the other oligonucleotide. Forsome applications, it may be desirable that the sequence of theoligonucleotide and its complimentary sequence is predetermined. Inother applications, the sequence may be unknown. A plurality ofdegenerate primers or primers with sequences predicted to bind to thecomplimentary sequence may be used. The primer-pair may be used toamplify a pair of complimentary sequences and the sequence between thetwo complimentary sequences of known or unknown size. In addition, theprimer may be used to amplify a template that is initiallydouble-stranded DNA or RNA, as well as single-stranded DNA or RNA. For asingle-stranded template, it is understood that the sequencecomplementary to one of the oligonucleotides will be synthesized byelongation of the first primer as it is annealed to its complementarysequence.

Primers for PCR or for reverse transcriptase initiated PCR may bepresent as a primer-pair, as is usual, or there may be only one primeras may be the case with genomic-sequencing or some protocols for reversetranscriptase initiated PCR.

In another embodiment of the invention, a component for subsequentanalysis may be a nucleotide sequence referred to as a "target sequencestabilizer." According to this embodiment, a "target sequencestabilizer" is a nucleotide sequence which is complimentary to an entireregion of RNA or DNA which is to be analyzed. A target sequencestabilizer may provide enhanced storage of an unstable single strand RNAor DNA stored as a sample of GM on the dry solid medium. As is known inthe art, many single strand RNAs are unstable and vulnerable todegradation by low levels of omnipresent RNases. However, the inventorbelieves that by including a target sequence stabilizer on the dry solidmedium, the target sequence stabilizer will bind to the single strandRNA (or DNA) sample and the "hybrid" formed is less vulnerable todegradation. For example, it is known in the art that double-strandedRNA/DNA hybrids are highly resistant to the common RNAases. The RNA orDNA annealed to the target sequence stabilizer may then be furtheranalyzed using methods known in the art, for example, LCR orcombinations of LCR and reverse transcriptase initiated PCR. Therefore,according to the invention, a component for subsequent analysis includesa component which functions by providing for protection from degradationof a stored sample of GM, such as a target sequence stabilizer.

The inclusion of a component for subsequent analysis, such as anucleotide sequence, provides a significant advantage for simplifyingsubsequent analysis of GM by, for example, automated PCR systems. Inaddition, inclusion of an appropriate nucleotide sequence may provideadditional protection from degradation of GM material such as unstableRNA.

Other components for subsequent analysis which may be included on thedry solid medium of the invention are, for example, ions, genetic probesor oligonucleotide or polynucleotide sequences with labels of diversetypes, and suitably stable enzymes such as thermo stable enzymes. Labelsmay include a fluorescent chemical group, radioactive isotope, chemicalbinding agent (e.g., biotin), antigens or enzymes.

The use of some wash systems for removal of protein or hemoglobinassociated with a sample of GM stored on a dry solid medium whichincludes a component for subsequent analysis may cause some loss of acomponent. The inventor has found that use of the single phase phenolwash of the invention to remove protein or hemoglobin associated with asample of GM on a dry solid medium which includes a nucleotide sequence,generally provides for reduced or insubstantial loss of the includednucleotide sequence. By "insubstantial loss" it is meant that the wash,when completed, has not caused sufficient loss of the component toinhibit subsequent analysis. In the case of a component primer,"insubstantial loss" means that the wash has not caused sufficient lossof primer to prevent PCR, LCR, reverse transcriptase initiated PCR,probing, etc. Hence, because both the nucleotide sequence and sample ofGM remain entrained to the dry solid medium during the washingprocedure, in situ processing is possible. One skilled in the art willreadily recognize the advantage this provides for analysis of GM usingautomated systems.

The loss of a component for subsequent analysis during processing of astored sample of GM may be advantageously overcome, in some washsystems, for example, aqueous wash systems, through the use of aretaining agent.

As used herein, a "retaining agent" is a compound to which a componentfor subsequent analysis may be added, mixed, suspended or otherwiseassociated with, to reduce loss of the component from the dry solidmedium during processing. A preferred retaining agent provides enhancedretention of a component to the dry solid medium when subjected toconditions which would otherwise cause greater loss of the componentfrom the dry solid medium. For example, a component for subsequentanalysis whose concentration on the medium could be reduced by using anonaqueous wash system may be combined with a retaining agent thatenhances retention of the component on the dry solid medium during anonaqueous extraction. A component for subsequent analysis whoseconcentration on the medium could be reduced by using an aqueous washsystem may be mixed with a retaining agent which enhances retention ofthe component on the dry solid medium during an aqueous extraction.

In one embodiment, the invention provides a retaining agent which may beassociated with a component for subsequent analysis when a sample of GMstored on the dry solid medium is to be processed using an aqueous washsystem. A preferred retaining agent will generally decrease the loss of,for example, a nucleotide sequence, when protein or hemoglobinassociated with a sample of GM stored on the dry solid medium isextracted using an aqueous wash system.

An example of a retaining agent for use with an aqueous wash system is awax. Alternatively, the retaining agent may be a hydrophilic carriersuspended in a wax.

As used herein, a "wax" is an organic mixture or compound of highmolecular weight which is solid at room temperature but liquifies athigher temperatures. Waxes include hydrocarbons, fatty acids, alcohols,esters, polymers, long chain amides, homologous silicon based compounds,and thermo-label resins.

In one preferred embodiment, a component for subsequent analysis, suchas a nucleotide sequence or an ion, is mixed with a hydrophilic solidcarrier and suspended in wax. Hydrophilic solid carriers include, forexample, starch, colloidal cellulose, cotton thread, and similarcompounds. The mixture of component and carrier may subsequently besuspended in a wax having a melting point at or below the temperature ofwhich subsequent analysis will occur. For example, if the sample of GMis to be analyzed using a PCR methodology a wax melting point of 45°C.-110° C., preferably 65° C.-90° C., should be selected. A preferredwax, according to the invention, is paraffin.

The wax suspension is allowed to solidify and the solid suspensiondeposited in small buttons or drops onto the dry solid medium. If aprimer is the component mixed with the wax suspension, each droppreferably contains sufficient primer for a single PCR amplification.Hence, as will be described infra multiple drops for multiple samples ofGM may be placed on a single dry solid medium card.

Although the inventor does not wish to be limited to a single theory, itis believed that during the aqueous system washing, the wax reduces theloss of the component to be retained. When, for example, the sample ofGM is analyzed using PCR methodology, the temperature rise during thefirst PCR cycle causes the wax to melt, thus releasing the component,such as a primer, to initiate the reaction.

In another example of a retaining agent for an aqueous wash system, acomponent for subsequent analysis may be added to a cotton threadcarrier and coated or impregnated with wax. For example, a cotton threadcould be drawn through a trough of an aqueous soluble component forsubsequent analysis. The thread could then be drawn through a dryingchamber followed by a trough containing hot wax. Rolls of the wax-coatedthread could be stored and subsequently cut into micro sections and heatstamped onto a dry solid medium to provide a component for subsequentanalysis on a cotton thread suspended in wax.

In another embodiment, a component for subsequent analysis may be mixedwith a wax without a hydrophilic carrier and applied to a dry solidmedium using xerography methodology. According to this embodiment, acomponent for subsequent analysis may be deposited onto the dry solidmedium in a water-insoluble retaining agent that becomes water solublewith the application of heat in the presence of water. Therefore, thecomponent for subsequent analysis may be mixed into a wax and can beprotected as a micro inclusion until the wax melts in water. Preferably,the wax containing a component for subsequent analysis are depositedonto predetermined locations on the dry solid medium. One example ofthis embodiment, is to apply hot, very dry wax dust containing acomponent for subsequent analysis onto the surface of the dry solidmedium, similar to xerography processes.

In another embodiment of the invention, a retaining agent may be solublein a wax using a hydrophilic liquid carrier. For example, a nucleotidesequence may first be made soluble in wax by complexing with adetergent. Preferred detergents are cationic detergents such ascetyltrimethyl ammonium bromide (CTAB). The detergent solubilizedcomponent is then mixed with wax and applied to the dry solid medium asdescribed above.

Hence, the use of a retaining agent, under certain conditions used toprocess a sample of GM, may enhance retention of a component forsubsequent analysis, thus yielding improved analytical results.

The advantage of including a component for subsequent analysis on thedry solid medium of the invention is readily seen in the case of PCRamplification of multiple samples using an automatic fluid delvingsystem. For example, by including a primer necessary for amplifying aspecific DNA or RNA gene sequence, multiple samples containing differentprimers may be analyzed using a single PCR reaction mixture. Typically,only one mixture of PCR enzyme and nucleotides to all vials isnecessary. Moreover, even if different primers included with differentGM samples require variation in reaction conditions such as magnesiumions and/or pH, these variations can be accommodated with only twoadditional pump or pipette systems to deliver inexpensive salts andbuffers that will cover most contingencies.

A component for subsequent analysis is preferably included on the drysolid medium prior to processing a sample of GM stored on the medium.Various permutations for inclusion of the components is possible. Forexample, the component may be added to the composition of the dry solidmedium prior to application of the composition to the solid matrix. Or,the components may be added to the solid matrix simultaneously with thecomposition. Alternatively, the composition of the invention may beapplied to the solid matrix and the components applied subsequently.Foreseeably, a sample of GM may be applied to a dry solid medium and acomponent for subsequent analysis applied to the dry solid medium aftercollection of the sample of GM but before processing the sample of GM.It is also foreseeable that a component may be added to a dry solidmedium after removing protein and/or hemoglobin but before subsequentanalysis.

Thus, the invention provides for novel and previously impracticalstrategies to be made feasible by a dry solid medium including acomponent for subsequent analysis in combination with methods that allowfor co-processing the component with the stored GM.

C. INCLUSION OF MULTIPLE NUCLEOTIDE SEQUENCES ON A SINGLE DRY SOLIDMEDIUM

The dry solid medium of the invention may also be identified or referredto as a card. A card may be prepared in such a manner as to providedifferent component for subsequent analysis at different locations on asingle card. When, for example, the component for subsequent analysis isa nucleotide sequence, multiple different nucleotide sequences forpriming PCR amplification or stabilizing different target sequences maybe included on one card.

As used herein, "multiple" means greater than one. A single sample of GMto be analyzed may be stored on a single card which is about 1 mm to 5mm square. Therefore, a card which is about 5 cm×10 cm could be divided,for example, into 200 individual samples. Each individual 5 mm squarecould hold a different nucleotide sequence. Accordingly, a single 5cm×10 cm card could provide for subsequent analysis of 200 differenttarget sequences.

A dry solid medium card provides for analysis of multiple regions of GMfrom one collected sample. The cards may be pre-made, pre-labelled andstored for future use. In one preferred embodiment, a nucleotidesequence included on a card for subsequent analysis may be a primer.According to this embodiment, all or many of the primers for aparticular application may be included on a single card. For example,one application may be to screen for multiple target sequences, in asingle GM sample, which together characterize a particular geneticdisease. Alternatively, a single card may contain primers to screen forthe genomes of multiple viral diseases. Hence, these cards, includingcomponents for subsequent analysis, may be prepared, labelled andpackaged for particular categories of usage, for example, penitentiarypurposes (e.g, individual identification), insurance company screeningand medical diagnosis of genetic diseases.

Moreover, the inclusion of, for example, a nucleotide sequence, allowsthe dry solid medium to be employed in different fields and still beconveniently processed together at one centralized, unspecialized centerfor automated analysis. This will allow the automated system to use onebasal mixture of enzyme, and possibly buffer, to simultaneously amplifyall target sequences for which primers are present on one type of card.As previously noted, the potential variations in ions and pH can behandled through a variety of ways, for example, separate delivery pumps.Alternatively, the use of a retaining agent may provide forindividualized delivery of ions or other components used in subsequentanalysis under varied conditions.

The following Examples are designed to teach those skilled in the arthow to practice the invention. They are not intended to define or limitthe scope of the invention in any way.

D. DRY SOLID MEDIUM KIT

The present invention also provides a kit for applying, storing orprocessing a sample of GM. A kit may include a dry solid medium and acontainer suitable for containing a dry solid medium during applying,storing or processing a sample of GM. According to this aspect of theinvention, the kit may include one or more containers. An example ofmore than one container which is suitable for a kit includes a 96 wellplate, as described below. The kit may also include one or more of anyof the dry solid media disclosed herein. This includes a dry solidmedium with or without a component for subsequent analysis and with orwithout a retaining agent. A dry solid medium included in a containermay be freely removable from the container or fixed within thecontainer.

The container of the kit may be any container suitable for use duringapplication of a sample of GM to the dry solid medium or duringapplication and one or more phases of subsequent processing of a sampleof GM. Therefore, a kit may be used to apply a sample of GM to a drysolid medium and the dry solid medium removed from the kit container forprocessing in a different container. Alternatively, a sample of GM maybe applied, stored, and processed all in the kit container.

One particularly useful aspect of a kit of the invention provides fortransfer of a liquid sample of GM from an original collection container(such as a blood tube) to a dry solid medium of the kit without humancontact. Hence, for example, using a robotic system such as a PLATO 3300II (Rosys, Rapperswil, Switzerland) a sample of whole blood can beremoved from an original collection container and transferred to a drysolid medium without need for human contact with the blood until afterit is dried on the dry solid medium. If the robotic system has trackingcapability, multiple samples of GM may be simultaneously transferred andtracked from original collection container onto dry solid medium in kitcontainers. For example, using a tracking system in which a bar code isplaced on a collection container and a bar code reader is present on therobotic system, the location of a sample from a particular collectioncontainer can be tracked through application of the sample of GM ontothe dry solid medium. Moreover, if the robotic system includes aprocessing system, a sample of GM may be removed from an originalcollection container, applied to the dry solid medium, and trackedthrough processing without need for human contact.

Advantages of a kit of the invention include: increased safety to humansample handlers during placement of a sample onto a dry solid medium,decreased human labor costs in applying a sample to the dry solidmedium, and decreased chance of sample contamination during handling.

A container for a kit of the invention may be made from any knownmaterial. The material of a kit container may be selected based on thetype of analysis to be performed on a sample of GM stored on a dry solidmedium. Typical materials from which a container of the kit may beprepared includes polystyrene, polypropylene and other materials used inthe art. A kit may include a single container with a dry solid medium.Alternatively, a kit may contain as many containers as a robotic orother transferring system can operate with. In a typical embodiment, akit of the invention may be a plate such as a Corning® 96-well assayplate (Corning Glass Works) or Falcon® 96-well assay plate (BectonDickenson and Company) with each well of the plate containing a drysolid medium. A multiple container kit such as a multi-well plate mayinclude a different dry solid medium in each container. Thus, forexample, a kit may be prepared with a single type of dry solid mediumfor screening multiple blood samples for a single gene whose primer maybe included on the dry solid medium. In another example, a kit may beprepared for screening a single blood sample for multiple genes. Thus,in this example, each container may contain a dry solid medium and eachdry solid medium include a different primer.

An example of using a kit with a 96 well container which can be used ona Rosys 3300 II to apply and wash a sample of GM in whole blood follows:

According to this example, each well of a 96 well plate, may include a 2mm to 5 mm, preferably about a 3 mm diameter, prepunched dry solidmedium disc in each of the 96 wells.

A 5 μl sample of whole blood is transferred from a blood collectiontube, pre-treated with EDTA, citrate, or heparin, onto a dry solidmedium disc in a well, using a new tip for each transfer. If the tubesare labeled with a bar code, and if the robotic system includes a barcode reader, each sample put onto a dry solid medium can be tracked. Theplate is incubated to dry the blood sample for about 10 to 20 minutes,preferably about 15 minutes at 45° to 55° C., preferably 50° C.Approximately 200 μl of Gentra One-Step™ (Gentra Systems Inc., Plymouth,Minn.) is added to each well and the plate is incubated forapproximately 10 to 20, preferably 15 minutes, at approximately 25° to30° C., preferably 27° C. This procedure is repeated two times. Afterthe second repeat, the wells are washed two times with approximately 200μl of 100% isopropanol. A well is then incubated for approximately 10 to20 minutes, preferably 15 minutes, at about 55° to 65° C., preferably60° C.

The dry solid medium containing a washed sample of GM in each of the 96wells may be removed if different subsequent analysis is to be performedon each sample. Alternatively, if all samples are to be subjected to thesame subsequent analysis, they may be further processed in the 96 wellcontainer.

EXAMPLES Example 1 In situ PCR Amplification of Blood DNA Stored on DrySolid Medium

A sample of blood DNA stored on the dry solid medium of the inventionwas amplified in situ by the polymerase chain reaction (PCR) technique.The dry solid medium used in this Example was cellulose based paperhaving sorbed thereto a composition comprising, per sq. cm of paper, 2micromols uric acid, 8 micromols tris (free base), 0.5 micromols EDTAand 1 mg SDS. The stored blood was washed and suitable ions added priorto PCR amplification.

1.1 Single Phase Phenol Wash

A. Materials

Solution A:

Single-phase phenol wash solution. A suitable mixture is phenol, 50 gmcontaining 120 mg of 8-hydroxyquinoline that has been saturated with 10ml of 1.0 M tris-acetate pH 8.0 and 1.0 ml 2-mercaptoethanol. Aftersaturation by shaking at room temperature, the aqueous phase isthoroughly removed and discarded.

Solution B:

Aqueous alcohol wash solution. 75% v/v Isopropanol, 25% v/v 0.1Mpotassium acetate at pH 7.8.

Solution C:

Ionic aqueous wash solution. 75% v/v Isopropanol, 25% v/v 0.01Mmagnesium acetate.

B. Method

All steps are preferably carried out in a single tube made of a suitablephenol resistant material, e.g. polyethylene.

1. Removal of protein: a 5 mm×5 mm square of blood impregnated dry solidmedium was treated with 1 ml of solution A, for approximately 1.5 hoursat 45° C. (since no primers were present, this temperature and time isnot critical). The solution A was then aspirated to waste and the papersquare quickly washed three times with 0.25 ml of solution A. Each washwas for only a few seconds and was immediately aspirated to waste.

2. Removal of phenol and addition of suitable ions: the dry solid mediumfrom step (a) above, was rapidly washed three times with 1 ml ofsolution B. Washes were at room temperature and were simple additionsfollowed by aspiration to waste. The paper was then washed for 20minutes at room temperature with solution C. (This is to saturate the GMon the paper with magnesium ions and remove the last of the phenol.) Thesolution C is aspirated to waste and the dry solid medium wassolvent-dried with one wash of pure isopropanol and then vacuum dried.

The washed dry solid medium was relatively quite white without anyobvious remnants of the red-brown color of blood. It was then used in aPCR reaction mix.

1.2 Amplification of DNA on Dry Solid Medium

The washed sample on dry solid medium described above has been shown tobe a suitable substrate for DNA polymerase chain reaction (PCR)amplification of DNA.

A. Materials

1. Specimens

Extracted DNA: 10 ml of blood was removed from a male volunteer usingstandard protocols.

Dry solid medium: Blood samples from the same volunteer were applieddirectly to the dry solid medium with subsequent single phase phenolwashing as described above. The dry solid medium was cut into about 1mm×1 mm pieces for use in PCR reactions.

2. Targets for Amplification

Target No. 1: Region of exon 2 of the n-Ras proto-oncogene onchromosome 1. The primers used are:

R1: 5' TGA CTG AGT ACA AAC TGG TGG TG 3' (SEQ ID NO:1)

R2: 5' CTC TAT GGT GGG ATC ATA TTC A 3' (SEQ ID NO:2).

The amplified DNA fragment obtained with these primers is 110 bp insize.

Target No. 2: A male specific Y chromosome repeat sequence. The primersare:

007: 5' TGG GCT GGA ATG GAA AGG AAT GCA AAC 3' (SEQ ID NO:3) and

008: 5' TCC ATT CGA TTC CAT TTT TTT CGA GAA 3' (SEQ ID NO:4). Theamplified DNA fragment obtained with these primers is 124 bp in size.

Target No. 3: A male specific Y chromosome repeat sequence. The primersused are:

004: 5' GAA TGT ATT AGA ATG TAA TGA ACT TTA 3' (SEQ ID NO:5) and

006: 5' TTC CAT TCC ATT CCA TTC CTT TCC TTT 3' (SEQ ID NO:6). Theamplified DNA fragment obtained with these primers is 250 bp in size.

B. Method

1. PCR Protocol

Extracted DNA (1 μg) or about 1 mm×1 mm fragments of washed dry solidmedium containing a sample of blood DNA were placed into 0.5 mlEppendorf tubes and made to 25 μl in PCR reaction mixture consisting of:

    ______________________________________                                        67      mM Tris HCl (pH 8.8 @ 25° C.)                                    16.6 mM (NH.sub.4).sub.2  SO.sub.4                                            2.0 mM MgCl.sub.2                                                             0.01% (w/v) gelatin                                                           0.1 mM deoxynucleotides (dATP, dTTP, dCTP, dGTP)                              0.25 μg of each primer (for respective target)                             0.25 U of Taq DNA polymerase.                                               ______________________________________                                    

2. The amplification conditions

The mixture was overlaid with 25 μl of light mineral oil and DNAamplification was performed by 30 cycles of amplification on a PerkinElmer-Cetus "thermal cycler". The first cycle consisted of:

    ______________________________________                                        DNA denaturation   6 min. @ 94° C.                                       Primer-DNA annealing 1 min. @ 55° C.                                   Taq DNA polymerase extension 1 min. @ 75° C.                         ______________________________________                                    

followed by 29 cycles as above except DNA denaturation was for 1 min @94° C. and the extension time on the last cycle was 10 min @ 72° C.before cooling the reaction mixture to 4° C.

1.3 Results

After amplification, 10 μl aliquots of PCR mixture were analyzed byelectrophoresis on 15% (w/v) polyacrylamide gels. Amplified target DNAwas visualized by UV illumination of the ethidium bromide stained gel.

An analysis of the products from PCR amplification of extracted bloodDNA and blood DNA stored on dry solid medium are shown in FIG. 1, asfollows:

Lanes 1-3: target No. 1, lane 1: 1 μg DNA, lane 2: 1 mm² filter, lane 3:control (no DNA);

Lanes 4-5: target No. 2, lane 4: 1 μg DNA, lane 5: 1 mm² filter; Lane 6:control (no DNA);

Lanes 7-9: target No. 3, lane 7: 1 μg DNA, lane 8: 1 mm² filter, lane 9:control (no DNA);

lane S: DNA size markers (pUC19/HpaII digest).

The results shown in FIG. 1 clearly demonstrate that the DNA has notbeen changed in any way as a result of its storage on the dry solidmedium of the invention, and that it can be used in situ as describedherein.

Example 2 PCR Reactions on DNA Stored on Dry Solid Medium IncludingPrimers

PCR analysis was performed on dry solid medium including primers addedto the dry solid medium prior to the collection of a sample of bloodDNA.

2.1 Genetic Material

A. Materials

1. DNA Templates

For preparing samples of genetic material containing DNA, 3 μl of humanblood was applied to a 3 mm square sample of dry solid medium, and theblood was allowed to dry. Unless otherwise indicated, this 3 mm squarewas then used in each PCR reaction. 3 μl of blood corresponds toapproximately 250 nanograms of white-cell DNA. In addition, a controlsample of purified human DNA in free solution was amplified using PCR.

2. Primers

Two synthetic oligonucleotide primers derived from the human Class IIHLA gene, DQ-alpha, were used in the experiments. Their sequences are:

5' GTGCTGCAGGTGTAAACTTGTACCAG 3' (SEQ ID NO:7)

5' CACGGATCCGGTAGCAGCGGTAGAGTTC 3' (SEQ ID NO:8)

The size of the PCR product expected using the two primers is 262 bp.Each primer was stored as a stock solution at a concentration of 50ng/ul. When added to the dry solid medium prior to processing, 150 ng ofboth primers was added to the 3 mm square of dry solid medium. Theprimers were always used together at equal concentrations.

2.2 Single Phase Phenol Wash

Solution A:

Single-phase phenol solution. 50 g of phenol, containing 120 mg of8-hydroxyquinoline, was saturated with 10 ml of 1.0 M Tris-acetate pH8.0 and 1.0 ml of 2-β-mercaptoethanol. After saturation at between 0° C.and 4° C., the aqueous phase was thoroughly removed and discarded.

Solution B:

Aqueous alcohol wash solution. 75% v/v isopropanol, 25% v/v 0.01Mpotassium acetate, pH 7.8.

Solution C:

Ionic aqueous wash solution. 75% v/v isopropanol, 25% v/v 0.01 Mmagnesium acetate.

B. Method

1. Washing the Dry Solid Medium

Samples of genetic material stored on the dry solid medium including aprimer were processed by first washing the medium with 200 μl ofSolution A for two hours at either 37° C. or 50° C., followed by twoquick rinses of the aqueous phase with Solution A. The medium was nextrinsed three times for five minutes each with 200 μl of Solution B atroom temperature. The medium was then washed with 200 μl of Solution Cfor 20 minutes at room temperature and then dried under vacuum. (Thewash times were all in excess. In practice, shorter times may be used.The effectiveness of the washes may be monitored by observing loss ofcolor: (1) the hemoglobin; and (2) the hydroxyquinoline.)

2.3 PCR Protocol

A. Materials

1. The Example PCR Mix 50 μl.

The following mixture was placed on washed dry solid medium containing asample of blood before heat-cycling:

    ______________________________________                                        67         mM tris-HC1, (pH 8.8 @ 25° C.)                                16.6 mM (NH.sub.4).sub.2  SO.sub.4  0.2 mg/ml gelatin                         0.2 mg/ml gelatin                                                             0.45% Triton X-100                                                            2.0 mM MgCl2                                                                  0.2 mM each of dATP, dTTP, dCTP, dGTP                                         2.5 units Taq polymerase.                                                   ______________________________________                                    

Template blood DNA was on dry solid medium or purified human DNA as freesolution. (50 ng template added when in free solution.)

As shown in table 1, primers were included with the dry solid mediumand/or in free solution. (100 ng each primer was if added as freesolution.)

B. Methods

1. The Amplification Conditions

4 min. @ 94° C. (start conditions)

1 min. @ 94° C.

40 sec. @ 50° C. (32 cycles)

1 min. @ 72° C.

4° C. (soak-end conditions)

The expected product is a 242 bp segment of the human class-II HLA gene,DQ-alpha.

2. Electrophoresis Conditions

Electrophoresis was in 1% agarose, 0.045 M tris, 0.045 M Boric acid, 1mM EDTA, pH 8.0, 1.5 hours at 8.5 volts/cm.

C. Results

3 mm×3 mm primer-loaded dry solid medium were taken. 3 μl of freshlycollected human blood from one donor was placed on all dry solid mediumsamples and allowed to dry. It was then stored at room temperature for 7days.

The squares of dry solid medium were then washed using the single phasephenol wash and all media-squares then dropped into PCR mixes eitherwith or without added primers. There were thus trials in which theprimers were added to the medium before loading blood to the medium,after loading blood to the medium, and to the final PCR mix (see Table1).

After the cycling 8 μl samples of each reaction mix were electrophoresedwith Promega PCR marker mix (Promega Corp., Madison, Wis.). (Bands at1000 bp, 750 bp, 500 bp, 300, 150, 50.) The gel was stained withethidium bromide and the product bands photographed (see FIG. 2).

Four samples were processed with the single phase phenol wash takingplace at 37° C., and four samples were processed with the single phasephenol wash taking place at 50° C. Subsequent processing steps and PCRreaction conditions for the two sets of samples were identical. FIG. 2shows that extractions performed at 37° C. generated bands of thepredicted size, and whose intensities varied (lanes 1-4), whileextractions performed at 50° C. yielded intense bands of the predictedsize (lanes 5-8). To show that the PCR products on DNA recovered fromthe dry solid medium were specific and corresponded to the expected PCRproduct, PCR reactions were performed on human DNA purified usingstandard methods (FIG. 2, lane 12), or using no DNA (FIG. 2, lane 13).No product was observed if no exogenous DNA was added to the reaction(FIG. 2, lane 13), and the product obtained with purified DNA comigratedwith the PCR product obtained from processed samples of medium (FIG. 2,compare lanes 11 and 12).

Two additional sets of samples were then extracted at 37° C. To onereaction primers were added to the dry solid medium after the blood. Toa second reaction primers were likewise added to the dry solid mediumafter the blood, and additional primers were added just prior to thebeginning of the PCR reaction. The results of this experiment are shownin FIG. 2, lanes 9 and 10, respectively. Lane 9 reveals that the DNA onthe dry solid medium generated a PCR product of the expected size whenprimers are added to the dry solid medium after the collection of theblood sample. A more intense PCR product of the expected size wasobtained when primers were added just prior to performing the PCRreaction (compare lanes 9 and 10). The results of these experiments aresummarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Experimental Conditions of PCR Reactions on DNA                                 Stored on Dry Solid Medium                                                                Time of                                                            Addition                                                                     Source of of Phenol Wash Lanes in Mean Results                                Template DNA Primers Temperature FIG. 2 of PCR                              ______________________________________                                        Human blood on                                                                          A        37° C.                                                                           1-4    ++                                          washed dry solid                                                              medium                                                                        Human blood on A 50° C. 5-8 ++++                                       washed dry solid                                                              medium                                                                        Human blood on A 50° C. 11 ++++                                        washed dry solid                                                              medium                                                                        Purified human D N.A. 12 ++++                                                 DNA in solution                                                               No DNA D N.A. 13 -                                                            Human blood on B 37° C.  9 +++                                         washed dry solid                                                              medium                                                                        Human blood on C 37° C. 10 ++++                                        washed dry solid                                                              medium                                                                      ______________________________________                                         KEY:                                                                          A = Primers included on dry solid medium before collection of blood           sample.                                                                       B = Primers included on dry solid medium after collection of blood on the     medium.                                                                       C = Primers included on dry solid medium after collection of blood on the     dry solid medium, and additional primers added just prior to PCR reaction     D = Primers added just prior to PCR reaction.                                 N.A. = Not applicable.                                                        ++++ = intense bands.                                                         +++ = Moderately intense bands.                                               ++ = Moderate bands.                                                          + = Weak bands.                                                          

In summary, the use of the dry solid medium containing primers isclearly practical, it opens a window of technique that makes large-scalePCR analysis much more practical and amenable to less skilled labor, andopens potential new applications, with unstable templates.

Example 3 In situ Digestion of Stored Blood with RestrictionEndonuclease Eco R1

To determine if samples of blood DNA stored on a cellulose-based drysolid medium were digestible in situ with restriction endonucleases, andthus suitable for procedures such as RFLP analysis, samples of bloodwere collected on cellulose-based dry solid medium, washed with singlephase phenol wash and digested with the restriction enzyme Eco R1. Inthis experiment, samples of blood were stored on the dry solid mediumfor as long as 18 months prior to processing.

3.1 Single Phase Phenol Wash

A. Materials and Methods

The single phase phenol wash and other wash solutions are described inExample 1 and 2. 1.5 ml of solution A was added to a dried 10 mm×10 mmpiece of dry solid medium containing approximately 0.1 ml of dried humanblood, and the solution was mixed using a standard laboratory mixer andincubated at 37° C. for 30 minutes. The solution A was then removed byaspiration and fresh solution A added. Treatments with solution A wererepeated until all the heme color disappeared from the dry solid medium.

Next, the samples were washed five times with solution B for two minuteseach, then twice with solution C, also for about two minutes each.Between each wash, the medium sample was subjected to centrifugalstripping. For this the medium was briefly spun in a small microfugetube in which a hole had been pierced and which had been nested inside alarger microfuge tube. The medium was then dried under vacuum or,alternatively, by centrifugal stripping. Following the processing stepsthe samples which initially were black after application of the bloodand subsequent drying, were white.

3.2 Digestion of Samples Stored on Dry Solid Medium Using RestrictionEnzymes

A. Material

Restriction enzyme Eco R1 were used in buffers supplied by BoehringerManheim.

B. Method

The washed dry solid medium was moistened with 50 μl of restrictionenzyme buffer containing an excess of restriction enzyme. For digestionswith the restriction enzyme Eco R1, 60 units of enzyme was added. Toensure complete wetting, the medium was wet in the buffer, centrifugallystripped, and remoistened with the buffer and enzyme mix. Digestionsproceeded for 2.5 hours at 37° C.

The restricted DNA was stripped from the medium by first centrifugallystripping the medium and collecting the stripped fluid. DNA was thenfurther removed by washing the solid medium with 25 μl of 7.5M ammoniumacetate, followed by a wash with 25 μl of 2.5M ammonium acetate. At eachwash step the medium was subjected to centrifugal stripping in order tomaximize recovery of the fluids. The fluids from the wash steps werecombined in a final volume of about 100 μl. The DNA was precipitatedfrom the collected fluid by adding 250 μl of ethanol, and air-drying thefinal pellet. The pellet was resuspended in 20 μl of water, and 10 μl ofthe resuspended pellet was used in gel electrophoresis.

3.3 Gel Electrophoresis

A. Conditions for Gel Electrophoresis

Digestion products were electrophoresed in a 1.5% agarose gel preparedin Tris-Acetate (composition was 0.01M Tris, 0.005M sodium acetate, 0.5MEDTA adjusted to pH 7.8 with acetic acid) at 50 V, 80 mA for 2 hours,after which the gel was stained with ethidium bromide and photographed.(See FIG. 3).

3.4 Results

To determine if blood samples stored on media of the invention forextended periods of time could be digested with restriction enzymes,samples of cellulose-based dry solid medium to which blood had beenadded 19 months previously, 11 months previously, or two weekspreviously were processed and then digested with Eco R1. FIG. 3 containsa photograph of the ethidium staining pattern and reveals a smearingpattern characteristic of an Eco R1 digest of human DNA. Thus, DNAstored on the medium for at least 19 months is digestible withrestriction endonucleases.

Example 4 Inactivation of Herpes Simplex Virus Type I and othermicroorganisms, by the Dry Solid Medium

In these experiments dry solid medium prepared according to theinvention was tested for its ability to inactivate herpes simplex virus,type I (HSV). HSV type I was chosen because it is a relatively resistantto inactivation and is transmissible through blood and other bodilyfluids.

4.1 Isolation of HSV Virus

A. Materials and Method

A recent clinical isolate of HSV type I recovered from an eye lesion wasgrown to high titre (>10⁸ plaque forming units/ml) in tissue cultureflasks of HEp2 epithelial and MRC-5 fibroblast cells, harvested off theflasks, and sonicated to release intracellular virus. Viral particleswere concentrated by high-speed centrifugation of a clarifiedsupernatant of the sonicate and were resuspended in viral transportmedium (VTM) containing 0.2M sucrose in a 0.02M phosphate buffer (pH7.6), 10% fetal calf serum, and antibiotics.

4.2 Application of Viral Particles to the Dry Solid Medium andSubsequent Recovery

For experiment I, the crude viral preparation was diluted 1:10 in eitherVTM or in fresh, heparinized blood. A 10 μl sample of the dilution wastested in duplicate on both untreated 10 mm×10 mm pieces of untreatedcellulose based paper and also on 10 mm×10 mm square pieces of dry solidmedium prepared according to the invention. After application of virus,the squares were supported in air by fine forceps, and after theappropriate time interval, the virus was eluted from the squares byvigorous agitation in 1 ml of MRC-5 maintenance medium (MM) (Eagles MEMbased medium with Earle's salts) containing 2% fetal calf serum.

In experiment II, a stock virus was taken from storage at -70° C. anddiluted 1:4 in citrated blood.

4.2 Assaying Viral Infectivity

A. Material and Method

The eluate was tested for infectivity using a fluorescent focus assay.Samples were diluted 1:100 in MM, and 0.2 ml of the dilution was thenadded to a well of MRC-5 fibroblasts in a 48-well cluster tray. After a45 minute incubation at 37° C., the sample was replaced with 0.5 ml MMand the tray was incubated overnight at 37° C. in the presence of 5%CO2. Cells were then fixed, stained with fluorescein-conjugated antibodyto HSV, and the number of fluorescent foci was counted under areverse-stage fluorescent microscope.

4.3 Results and Discussion

HSV type 1 viral particles were added either to untreated cellulosebased paper or to cellulose based paper treated according to theinvention to produce the dry solid medium, and then eluted after varyinglengths of time. In one experiment, the virus was stored in VTM, and inthe second the virus was resuspended in blood. The results of theexperiment are shown in Table 2. In both experiments, viral infectivitywas reduced to undetectable levels--in these conditions, greater than99.5% inhibition--when virus was added to the dry solid medium, evenwhen the viral particles were eluted immediately. Viruses in bloodeluted from untreated cellulose based paper (CBP), in contrast, wereonly slightly, if at all, inhibited. A similar inhibitory effect on HSVinfectivity was observed when the experiments were performed withviruses recovered from a frozen stock.

The invention has also shown the inhibitory effect of the dry solidmedium on growth of Staphylococcus aureus, Staphylococcus epidermidisand adventitious microorganisms in the environment.

                  TABLE 2                                                         ______________________________________                                        Infectivity of HSV Following Application on to                                  Untreated cellulose based paper (CBP) Paper or on                             to Dry Solid Medium                                                                  A. HSV in VTM   B. HSV in Blood                                               Viral     Per Cent          Per Cent                                   Treatment Titer Inhibition Viral Titer Inhibition                           ______________________________________                                        Untreated                                                                              1.9 × 10.sup.7                                                                    0                 --                                         (virus added                                                                  directly to                                                                   MM)                                                                           Virus spotted <5.0 × 10.sup.4  >99.5 <5 × 10.sup.4  99.5                                              on treated CBP (no virus  (no virus       and detected)  detected)                                                      immediately                                                                   eluted into MM                                                                Virus spotted 1.2 × 10.sup.7 37  9.8 × 10.sup.6 48                on untreated                                                                  CBP and                                                                       immediately                                                                   eluted into MM                                                                Virus spotted  <5 × 10.sup.4 >97  <5 × 10.sup.4  >97                                                  on treated CBP (no virus  (no virus       and eluted detected)  detected)                                               onto MM after                                                                 10 minutes                                                                    Virus spotted 1.8 × 10.sup.6 90 9.7 × 10.sup.6 47.5                                                   on untreated                              CBP and eluted                                                                into MM after                                                                 10 minutes                                                                  ______________________________________                                    

Example 5 Method for treating the dry solid medium of the invention foruse in standard analysis systems

The dry solid medium of the invention, with or without components forsubsequent analysis can also be used in standard assays of analytes fordiagnostic purposes, e.g., in the Guthrie Test for phenylketonuria (PKU)and enzymic assays such as a test for galactosemia. Both of these testsare routinely carried out on newborn infants. Prior to analysis usingsuch standard assay systems, it is preferable to neutralize the SDS andEDTA on the dry solid medium containing a sample of blood before theanalysis of such analytes by adding a "converter solution".

5.1 Dry Solid Medium Converter Solution

The dry solid medium converter solution works by binding the SDS andEDTA.

A. Materials and Methods

The dry solid medium converter solution includes the following mixture:

1. 577 mg Protamine sulphate (salmine) Sigma No-4380, lot 71F-0037, isadded to 7.69 ml water and shaken (to give 75 mg/ml).

This batch will be nominally 75 mg/ml before dialysis. Thisconcentration is near to the solubility of the protamine. It dissolvesslowly as a gluey turbid mass.

2. Dialysis: this is a clean-up precaution to assure that the materialis essentially sterile and fully dissolved.

The gluey turbid mass is placed in a dialysis bag approx. 1.5 cm indiameter by 2 cm to 10 cm in length. Both ends of the bag are knottedtightly. The bag is then weighed and dropped into a 1 liter flaskcontaining about 800 ml of 50 mM of magnesium acetate with chloroformand a stirring bar. The material is dialyzed in the flask, overnight,against the 800 ml of 50 mM magnesium acetate with an excess ofchloroform (approx. 5 ml) present as a sterilizing agent. The protaminealmost completely dissolves overnight giving a clear solution and fewobvious small lumps of debris.

The weight of the bag increases:

Weight of bag before dialysis =8.1 g 20 hour dialysis, 4° with CHCl₃saturated fluid.

Weight of bag after dialysis=8.67 g.

Thus, the concentration of protamine is now, nominally, 70 mg/ml.(Assuming no losses through bag.)

3. The bag was then punctured at the bottom, and the fluid collected andcentrifuged at 4000 rpm (approx. 2000×g) for 10 mins. to remove debris.

4. 1 ml lots were stored at -80° C.

5. Prior to analysis, about 3 ul of the converter solution is applied toeach 3 mm diameter disk of dry solid medium containing a sample ofblood.

5.2 PKU Screening Procedure (Guthrie Test)

The Guthrie test is based on the stimulatory effect of phenylalanine onthe growth of Bacillus subtilis in the presence of beta-2-thienylalaninegrowth inhibitor.

Disks of blood samples stored on the dry solid medium of the inventionare placed on agar containing bacterial spores and the inhibitor.Following incubation at 37° C. for 16 to 18 hours, the diameter of thegrowth around the disk is proportional to the concentration ofphenylalanine. Higher than normal levels in infants is indicative ofphenylketonuria.

An advantage of using the dry solid medium for the Guthrie test is thatthe disk of dry solid medium retains proteins and hemoglobin from thesample, leaving a cleaner area around the disk and making theinterpretation of the result easier. In fact, samples of bloodcontaining phenylalanine which were stored on the dry solid mediumconsistently gave at least as strong bacterial growth rings asequivalent samples on standard paper.

Also, because sufficient bacterial spores are used in a Guthrie test andthe rate of growth is so fast, the inhibition or killing of bacteria byreagents of the dry solid medium which were proximal to the dry solidmedium did not cause a problem for interpreting the results. Therefore,when using the dry solid medium it is not necessary to apply theconverter solution to the disk prior to the test. However, it is anadvantage to do this to remove an observable weak zone of growthinhibition.

5.3 Screening for Galactosemia

Galactosemia screening requires the use of active enzymes to assay forgalactose and galactose phosphate. The main enzyme, beta-galactosedehydrogenase reduces NAD to NADH and this is observedspectrophotometrically.

This assay is severely inhibited by components of the composition of thedry solid medium, but the inhibition can be substantially eliminated bythe application of converter solution to a sample of blood stored on thedry solid medium.

After addition of the converter solution, just prior to the enzymicassay, the assay has been observed to perform in the same way as it doeswith a sample of blood on untreated cellulose based paper. Of course,the dry solid medium has the advantage of providing the benefits ofpathogen killing and preservation of the sample compared to standardmaterials used.

All patents in the specification are indicative of the level of ordinaryskill in the art to which this invention pertains. All patents areherein incorporated by reference to the same extent as if eachindividual patent and publication was specifically and individuallyindicated by reference.

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made in the invention without departingfrom the spirit or scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 8                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TGACTGAGTA CAAACTGGTG GTG           - #                  - #                    23                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CTCTATGGTG GGATCATATT CA           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TGGGCTGGAA TGGAAAGGAA TGCAAAC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TCCATTCGAT TCCATTTTTT TCGAGAA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GAATGTATTA GAATGTAATG AACTTTA          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - TTCCATTCCA TTCCATTCCT TTCCTTT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GTGCTGCAGG TGTAAACTTG TACCAG          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CACGGATCCG GTAGCAGCGG TAGAGTTC         - #                  - #                 28                                                                    __________________________________________________________________________

What is claimed is:
 1. A dry solid medium for storing at least onesample of genetic material, said dry solid medium comprising:(a) a solidmatrix having sorbed thereto a composition comprising a proteindenaturing agent; (b) a component for subsequent analysis of said sampleof genetic material; and (c) a retaining agent for retaining saidcomponent for subsequent analysis on said solid matrix, wherein saidretaining agent comprises a hydrophilic solid carrier in wax and whereinsaid hydrophilic solid carrier comprises a starch, cellulose or cottonmaterial.
 2. The dry solid medium according to claim 1, wherein saidprotein denaturing agent is a detergent.
 3. The dry solid mediumaccording to claim 1, wherein said sample of genetic material stored onsaid dry solid medium is RNA.
 4. The dry solid medium according to claim1, wherein said sample of genetic material stored on said dry solidmedium is blood DNA.
 5. The dry solid medium according to claim 1,wherein said component for subsequent analysis of said sample of geneticmaterial is a nucleotide sequence.
 6. The dry solid medium according toclaim 1, wherein said component for subsequent analysis of said sampleof genetic material is a target sequence stabilizer.
 7. The dry solidmedium according to claim 1, wherein said retaining agent reduces lossof said component for subsequent analysis during an aqueous washprocedure.
 8. The dry solid medium according to claim 1, wherein saidcomponent for subsequent analysis of said sample of genetic material isapplied to said dry solid medium after said sample of genetic materialis applied to said dry solid medium.
 9. The dry solid medium accordingto claim 1, wherein said dry solid medium is a card comprising more thanone component for subsequent analysis.
 10. The dry solid mediumaccording to claim 1, wherein said composition of said dry solid mediumfurther comprises a free radical trap.
 11. The dry solid mediumaccording to claim 2, wherein said detergent is an anionic detergent.12. A method for storing and subsequent analysis of a sample of geneticmaterial comprising:(a) applying a sample of genetic material to a drysolid medium, said dry solid medium comprising:(i) a solid matrix havingsorbed thereto a composition comprising a protein denaturing agent; (ii)a component for subsequent analysis of said sample of genetic material;and (iii) a retaining agent for retaining said component for subsequentanalysis on said matrix, wherein said retaining agent comprises ahydrophilic solid carrier in wax and wherein said hydrophilic solidcarrier comprises a starch, cellulose or cotton material; (b) storingsaid sample of genetic material; (c) washing said sample of geneticmaterial; and (d) analyzing said sample of genetic material.
 13. Themethod according to claim 12, wherein any of said steps (a), (b), (c)and (d) are performed on an automated system.
 14. The method accordingto claim 12, wherein said sample of genetic material analyzed is RNAwhich is first transcribed to DNA using reverse transcriptase initiatedpolymerase chain reaction.
 15. The method according to claim 12, whereinsaid sample of genetic material of step (d) is analyzed using polymerasechain reaction.
 16. The method according to claim 12, wherein saidwashing of said sample of genetic material of step (c) is performedusing an aqueous wash system.
 17. A method for storing a sample ofgenetic material comprising:(a) applying a sample of genetic material toa dry solid medium, said dry solid medium comprising:(i) a solid matrixhaving sorbed thereto a composition comprising a protein denaturingagent; (ii) a component for subsequent analysis of said sample ofgenetic material; and (iii) a retaining agent for retaining saidcomponent for subsequent analysis on said solid matrix, wherein saidretaining agent comprises a hydrophilic solid carrier in wax and whereinsaid hydrophilic solid carrier comprises a starch, cellulose or cottonmaterial; and (b) storing said sample of genetic material.